The development of medicines that can efficiently treat intractable diseases, such as cancer and AIDS, is one of the most important objects in the life science field. One of the methods that has high potential as a solution is the use of gene medicines that acts only on specific genes. Particularly, an RNA interference (RNAi) method using a 21-base-long, short double-stranded RNA (small interfering RNA: siRNA) has been attracting attention recently. The RNAi method was first reported by Fire et al. in 1998 (see Non-Patent Document 1). According to the method reported by Fire et al., an approximately 100 base pair double-stranded RNA that is homologous to a specific region of a gene whose function is to be inhibited is introduced into a cell and digested into about 20- to about 25 base pair double-stranded RNA fragments by the action of Dicer in the cytoplasm. The RNA fragments are then combined with a plurality of proteins to form an RNA/protein complex (this complex is referred to as an “RISC”: RNA-Induced Silencing Complex). This complex binds to a homologous region of mRNA produced from the target gene, thereby potently suppressing the gene expression. However, it is reported that when approximately 30 base pair or longer double-stranded RNA is introduced into mammalian cells, an interferon response that is an antiviral response is induced, thus causing the phenomenon of apoptosis. It was thus considered difficult to apply the RNAi method to mammalian cell systems. In view of this problem, Tuschl et al. chemically synthesized a 21-base-long double-stranded RNA that has dangling ends at both 3′ ends of the strands, and reported that direct introduction of the 21-base-long double-stranded RNA into mammalian cells can sequence-specifically and potently suppress gene expression, while avoiding an interferon response (see Non-Patent Document 2). Tuschl et al. further synthesized short double-stranded RNAs in which the double-stranded region is 19 base pairs and has dangling ends of varied lengths at the 3′ ends or 5′ ends, and investigated their RNA interference effects. The results showed that 21-base-long siRNA having 2-base-long dangling ends at both 3′ ends produces a very high RNA interference effect, whereas no other types of short double-stranded RNAs produce a remarkable RNA interference effect. Based on this report, the principal method used today is an RNA interference method using a 21-base-long double-stranded RNA having 2-base-long dangling ends at both 3′ ends. The method for inhibiting the expression of a target gene using a 21-base-long, short double-stranded RNA is herein referred to as the “siRNA method”, to distinguish it from the RNAi method.
Because the siRNA method uses synthetic RNA, sample preparation is comparatively easy, and handling is also easy. Furthermore, very potent effects can be produced. Therefore, the siRNA method has been attracting much attention not only in the life science field, but also in the biotechnology business sector.
However, there are also problems with this excellent siRNA method that must be solved. As described above, siRNA is composed of RNA molecules that are readily digested by the action of nuclease contained in cells or in a medium. Although the double-stranded RNA region has a relatively high resistance to nuclease compared to single-stranded RNA, 19 base pair double-stranded RNA hardly exhibits an RNA interference effect at conventional levels. As such, it has been reported that although synthetic siRNA exhibits a potent suppressive effect on gene expression for about 2 to about 4 days after introduction into cells containing a target gene sequence, its RNA interference effect is sharply reduced thereafter, and is almost completely lost in about 7 days.
Various chemically modified siRNAs have recently been disclosed with the purpose of achieving a high cellular uptake efficiency and a prolonged, highly active RNA interference effect in synthetic RNA. For example, siRNAs terminally modified with an amino group, a thiol group, or an abasic site have been synthesized to enhance resistance to exonuclease digestion. However, it has been reported that terminal modification of 21-base-long siRNA sharply reduces the RNA interference effect in most cases.
A recent report by J. Rossi et al. revealed that a 27 base pair double-stranded RNA has an RNA interference effect that is about 100 times greater than that of a 21-base-long siRNA (see Non-Patent Document 3). This potent effect is considered to be achieved for the following reason: after a 27 base pair RNA is cleaved with Dicer, which is an RNase III-like enzyme, into a 21-base-long siRNA, the siRNA is recognized as is by the protein complex RISC, so that siRNA effects can be produced with high efficiency.
Since 27-base-long RNA can produce an excellent RNA interference effect as described above, expectations for its future use as a gene medicine have been increasing. However, what technical method is useful for further enhancing the RNA interference effect of the 27-base-long RNA is completely unknown. Furthermore, the technical method for enhancing the RNA interference effect of double-stranded RNAs of base lengths other than 27 bases is also unknown.
Double-stranded RNAs that produce an RNA interference effect are usually structured to have one or more dangling ends at the ends. The RNA interference effects of double-stranded RNAs having no dangling end (i.e., blunt-ended) have also been investigated. However, the results suggested that double-stranded RNAs that are blunt-ended on the 5′-end side of the sense strand has an RNA interference effect that is substantially the same as or lower than that of double-stranded RNAs having a dangling end on the 5′-end side of the sense strand (see Non-Patent Document 4).
Lipids have a high affinity to cell membranes and high permeability through cell membranes, and are known to be useful for delivering drugs into cells. Binding such a lipid to a double-stranded RNA that has an RNA interference effect would be expected to enhance the cellular uptake efficiency and increase the RNA interference effect. However, merely binding a lipid to a double-stranded RNA having an RNA interference effect has been known to sharply reduce the RNA interference effect. In the prior art, a lipid-modified RNA that can produce both an excellent RNA interference effect and a useful effect based on the lipid has yet to be constructed.
Non-Patent Document 1: Fire et al., Nature, 391, 806-811 (1998)
Non-Patent Document 2: Tuschl et al., EMBO Journal, 20, 6877-6888 (2001)
Non-Patent Document 3: J. Rossi et al., Nature Biotech., 23, 222-226 (2005)
Non-Patent Document 4: J. T. Marques et al., Nature Biotech., 24, 559-565 (2006).